The present invention relates to novel mammalian phospholipase A2 nucleotide sequences, low molecular weight (Approximately 14 KD) amino acid sequences encoded thereby, clones and vectors which include the mammalian phospholipase A2 nucleotide sequences, antisense nucleotide sequences complementary to the genes and mRNA transcripts encoding for the phospholipase amino acid sequences, nucleotide sequences having internal ribosome binding sites which allow for internal initiation of mRNA cap-independent translation, and cell lines.
Phospholipase A2sxe2x80x94phosphatide 2-acyl-hydrolase, EC 3.1.1.4 (hereinafter xe2x80x9cPLA2xe2x80x9d) constitute a diverse family of enzymes that hydrolyze the sn-2 fatty acyl ester bond of phosphogylcerides, producing free fatty acid and lysophospholipids. See Dennis, E. A. Phospholiphases. In: The Enzymes, edited by Boyer, P. New York: Academic Press, p. 307-353 (1983). Over the past two decades, PLA2 activities have been purified and characterized from different tissues, cultured cells, and exudates from different mammals. See Rordorf, G. et al.: J. Neuroscience, 11:1829-1826 (1991); Seilhamer, J. J. et al.: J. Biochem., 106:38-42 (1989); Langlais J. et al.: Biocham. and Biophys. Res. Comm., 182:208-214 (1992); Murakami, M. et al.: J. Biochem., 111:175-181 (1992); and Jordan, L. M. et al.: J. Chromat., 597:299-308 (1992). These enzymes have been found to vary in molecular weight, optimal pH, Ca2+ dependence, substrate specificity, and solubility.
To date, two classes of unrelated PLA2s have been reported. One is a family of low molecular mass, approximately 14 kDa PLA2s which are characterized by a rigid three dimensional structure maintained by disulfide bridges and a catalytic requirement for Ca2+. The other is a high molecular mass, 82 kDa, intracellular PLA2 found in the cytosolic subcellular fraction in the absence of calcium, but associated with cellular membranes at calcium concentrations from 0.1 to 10 xcexcM. See Clark, J. D. et al.: Cell, 65:1043-1051 (1991) and Sharp, J. D. et al.: J. Biol. Chem., 266:14850-14853 (1991). In addition, several Ca++-insensitive PLA2 activities are believed to exist, however, it is also believed that as yet none of the genes encoding such activities have been cloned.
In terms of structure, low molecular weight, e.g., about 14 kDa, PLA2s rank among the best characterized enzymes. Complete primary sequences have been determined for more than 50 PLA2s from organisms such as snakes, bees and humans. See Heinrikson, R. L.: Methods in Enzymology, 197:201-214 (1991); Davidson, F. F. et al.: J. Mol. Evolution, 31:228-238 (1990); and Dennis, E. A. Phospholiphases. In: The Enzymes, edited by Boyer, P. New York, Academic Press, p. 307-353 (1983). In all active 14 kDa PLA2s, 18 amino acids are believed to be conserved. See Heinrikson, R. L.: Methods in Enzymology, 197:201-214 (1991) and Davidson, F. F. J. Mol. Evolution, 31:228-238 (1990). Based on selected structural determinants, the low molecular weight PLA2s have been classified into two types. See Heinrikson, R. L. et al.: J. Biol. Chem., 252:4913-4921 (1977). Type I enzymes have a disulfide bridge connecting cysteines between amino acids 11 and 77. In addition, there is an insertion of three amino acids between residues 54 and 56, the so-called elapid loop. The only identified mammalian Type I PLA2s, see Seilhamer, J. J. et al.: DNA, 5:519-527 (1986) and Ohara, O. et al.: J. Biochem., 99:733-739 (1986), are expressed mainly in the pancreas and function extracellularly in digestion. Type II PLA2s, on the other hand, lack the disulfide bridge between amino acids 11 and 77, have carboxy-terminal (COOH-terminal) amino acid extensions which can vary in length, but are commonly seven amino acids in length, which typically terminate in a half-cysteine joined to Cys-50 near the catalytic site His-48. The mammalian Type II PLA2s reported to date occur in trace amounts in several tissues such as liver and spleen and are secreted from various cells in response to appropriate stimuli. See Seilhamer, J. J. et al.: J. Biol. Chem., 264:5335-5338 (1989); Kramer, R. M. et al.: J. Biol. Chem., 264:5768-5775 (1989); Komada, M. et al.: J. Biochem., 106:545-547 (1989); Kusunoki, C. et al.: Biochimica Et Biophysica Acta, 1087:95-97 (1990); Aarsman, A. J. et al.: J. Biol. Chem., 264:10008-10014 (1989); Ono, T. et al.: J. Biol. Chem., 264:5732-5738 (1988); Horigome, K. et al.: J. Biochem., 101:53-61 (1987) Nakano, T. et al.: Febs. Letters, 261:171-174 (1990); and Schalkwijk, C. et al.: Biochem. and Biophys. Res. Comm., 174:268-272 (1991). It is believed that Type II PLA2s are associated with the pathologies of several diseases involving infection, tissue damage, and inflammation, such as acute pancreatitis, septic shock, peritonitis and rheumatoid arthritis. See Vadas, P. et al.: Lab. Invest., 55:391-404 (1986); Pruzanski, W. et al.: Advances in Exper. Med. and Biol., 279:239-251 (1990); Uhl, W. et al.: J. Trauma, 30:1283-1290 (1990); and Malfertheiner, P. et al.: Klinische Wochenscrift, 67:183-185 (1989). Mammalian Type I and II PLA2s share approximately 30-40% amino acid homology; however, eighteen amino acids are invariantly conserved in all known functional PLA2s. Type I mammalian PLA2 genes contain 4 coding exons; Type II mammalian genes contain five exons, the first of which is noncoding.
In 1990, a distinct 120 bp putative PLA2 exon-like fragment (h10a), homologous to the amino-terminus encoding region of known PLA2s, was obtained by screening a human genomic DNA library with a 45 bp human PLA2 Type II oligonucleotide probe. See Johnson, L. K. et al.: Advances in Exper. Med. and Biol., 275:17-34 (1990). Zoo blots indicated that the putative exon has been highly conserved during evolution. However, additional exons indicating the presence of a complete gene, a corresponding cDNA, or evidence of transcription in different human tissues was not found.
Neuronal ceroid lipfuscinoses (NCL), or Batten disease, are terminal, inheritable, lysosomal storage diseases of children. They are characterized by the accumulation of an autofluorescent pigment (ceroid or lipofuscin) in cells, especially neurons and epithelial pigment cells of the retina. NCL patients typically manifest high levels of the highly reactive compound, 4-hydroxynonenal. These levels are believed to be a consequence of a failure to resolve peroxidized, fatty acids in the normal way. It is believed that this failure could be the result of a phospholipase A2 defect.
The infantile form of NCL has now been linked to chromosome 1p33-35. See Jarvela, I. et al.: Genomics, 9:170-173 (1991). The non-pancreatic PLA2 (Type II) has also been mapped to chromosome 1. The Type II gene and two additional putative exon-like xe2x80x9cfragmentsxe2x80x9d (h8 and h10a), see Johnson, L. K. et al.: Advances in Exper. Med. and Biol., 275:17-34 (1990), are located at about 1p34xe2x80x94in the middle of the region where gene for infantile NCL is believed to reside. See Jarvala, I. et al.: Genomics, 9:170-173 (1991). h8 and h10a each contain a unique sequence which is highly homologous to, but distinct from, exon two (which contains the calcium binding domain) of PLA2 Type II.
Consequently, there is a continuing need to further identify and characterize additional PLA2 exons if such exist. Such exons could be part of unidentified genes. To the extent there are additional unidentified PLA2 exons and genes, they may encode proteins (enzymes) that function in a manner different from, similar to, or overlapping with, the known PLA2s. Moreover, such unidentified exons and/or genes and the enzymes encoded thereby may be regulated by some of the same effectors as the known PLA2 genes and their proteins. Investigation of these unidentified exons and/or genes and their encoded enzymes may therefore result in new approaches to therapy of PLA2-related diseases, such as Batten disease and inflammatory disease. Alternatively, these unidentified enzymes may have entirely different physiologic and pathologic functions. Thus, therapeutic approaches designed to block the activity of the known Type II PLA2 enzymes may also block or reduce the activity of these novel enzymes, thereby producing unexpected side effects. Still further, a better understanding of the regulation of expression of the known and unidentified Type II PLA2 genes in different tissues will likely expand the overall understanding of the biology and metabolic processes involving PLA2s.
In brief, the present invention overcomes certain of the above-mentioned shortcomings and drawbacks associated with the present state of the PLA2 art through the discovery of a novel family of mammalian PLA2 genes or nucleic acid sequences encoding low molecular weight amino acid sequences, clones, vectors, antisense nucleotide sequences, nucleotide sequences having internal binding sites, and cell lines.
More particularly, the low molecular weight, i.e., about 14 kDa, amino acid sequences encoded by the novel family of mammalian PLA2 genes or sequences of the present invention may be generally characterized as enzymes having esterase activity specific for the acyl group at the sn2 position of glycero-phospholipids. Moreover, the novel amino acid sequences of the present invention do not include disulfide bridges between cysteine amino acids 11 and 77 and elapid loops. Still further, the novel amino acid sequences of the present invention may in some instances include COOH-terminal amino acid extensions which can vary in length. In addition, because of the difference in the number of cysteine residues in the encoded amino acid sequences, those novel PLA2s of the present invention that include 16 cysteine amino acid residues have been designated as Type III whereas those novel Type IV PLA2s of the instant invention include 12 cysteines and have been designated at Type IV. Exemplary of Type, III PLA2s of the present invention are the genes identified as RPLA2-8 (rat) and partial HPLA2-8 (human, as well as the RPLA2-8 (rat) cDNA. Examples of Type IV PLA2s of the present invention are the cDNAs identified as RPLA2-10 (rat) and HPLA2-10 (human).
In accordance with the present invention, a human PLA2-encoding cDNA, which expresses HPLA2-10, see FIG. 12, has been isolated from human brain RNA by RACE-PCR technique. The HPLA2-10 cDNA also has been isolated from a human stomach cDNA library. In addition, two rat PLA2 encoding cDNAs, designated RPLA2-8 (FIG. 3) and RPLA2-10 (FIG. 11), have been isolated from rat brain and heart cDNA libraries, respectively. The RPLA2-10 is believed to be the counterpart of the HPLA2-10. RPLA2-10 and HPLA2-10 share about 79% and 78% homology at the open reading frame nucleic acid and amino acid sequence levels, respectively. The mature enzyme encoded by the HPLA2-10 clone has a calculated molecular weight of about 13,592, whereas the mature enzyme encoded by the RPLA2-8 clone has a calculated molecular weight of about 14,673. As indicated, a partial human genomic counterpart to RPLA2-8, HPLA2-8 genomic DNA, has been isolated. See FIG. 19.
Comparison of the RPLA2-8 amino acid sequence deduced from the cDNA sequence to Type I and Type II PLA2s is shown in FIGS. 8 and 9. The significant structural features of the RPLA2-8 protein are summarized in TABLE I. Seventeen (17) of the eighteen (18) absolutely conserved amino acids in all active 14 kDa PLA2s are conserved in RPLA2-18. RPLA2-8 protein does not contain either a disulfide bridge between Cysteines 11 and 77 or an elapid loop, which are both characteristic of Type I PLA2s. RPLA2-8 protein, however, does include a seven amino acid COOH-terminal extension having the sequence GRDKLHC, as shown in FIG. 27, which is a characteristic of Type II PLA2s as evidenced in FIGS. 22 and 27. Furthermore, unlike mammalian type I and II PLA2s which have 14 cysteine amino acid residues, RPLA2-8 protein includes 16 cysteine amino acid residues. It is therefore believed that RPLA2-8 encodes a novel PLA2, which has been designated as PLA2 Type III.
The cDNAs of RPLA2-10 and HPLA2-10 are 1.8 kb (FIG. 11) and 1.1 kb (FIG. 12), respectively. A comparison between the deduced amino acid sequences from RPLA2-10 and HPLA2-10 is shown in FIG. 13. FIGS. 14 and 15 are comparisons between the HPLA2-10 deduced amino acid sequence and those of Type I and II human PLA2s, respectively. FIGS. 18 and 16 are comparisons between the RPLA2-10 deduced amino acid sequence and those of Type I and II rat PLA2s, respectively. A comparison between the deduced amino acid sequences from RPLA2-10 and RPLA2-8 is shown in FIG. 17. The major structural features of human and rat PLA2-10 deduced amino acid sequences are listed in TABLE I. All eighteen (18) conserved amino acids in all of the active low-molecular weight, approximately 14 kDa, PLA2s are conserved in both human and rat PLA2-10 amino acid sequences of the present invention. Like the predicted RPLA2-8 amino acid sequence, human and rat PLA2-10 amino acid sequences also lack disulfide bridges between Cys-11 and 77 and elapid loops. However, PLA2-10 amino acid sequences are believed to differ from RPLA2-8 protein by having twelve (12) cysteine residues instead of sixteen (16). They further differ from RPLA2-8 in that RPLA2-10 does not have a COOH-terminal amino acid extension as depicted in FIG. 27 and HPLA2-10 has only a single serine amino acid COOH-terminal extension as illustrated in FIG. 22. The PLA2-10 proteins of the present invention have therefore been designated, as mentioned hereinbefore, as PLA2 Type IV.
The present invention also contemplates antisense nucleotide sequences which are complementary to the genes and mRNA transcripts which encode for the Type III and Type IV PLA2s. Exemplary of antisense sequences in accordance with the present invention are those which are complementary to the entire or portions of the nucleotide sequences set forth in FIGS. 3, 11, 12 and 19. It should therefore be understood that the present invention contemplates any antisense nucleotide sequence which may be useful in connection with inhibiting or interfering with the expression of the Type III and Type IV PLA2 enzyme genes and mRNA transcripts therefor.
The above features and advantages will be better understood with reference to the FIGS. Detailed Description and Examples which are set out hereinbelow. It should be understood that the biological materials of this invention are exemplary only and are not to be regarded as limitations of this invention.